IP Troubleshooting: Co-IP is Not Successful

In some cases, co-IP is not successful for finding and/or confirming protein interactions. Co-IP is most useful for high-affinity interactions. If low-affinity or transient interactions shall not be detected, co-IP often fails without further optimization such as crosslinking.
In some cases, lysis conditions simply cannot be optimized in a way to allow efficient extraction of proteins while retaining their binding properties. However, there are a number of alternate techniques that you can choose, such as Pull-Down assays (similar to co-IP), but bait protein of interacting pair is immobilized instead of antibody (bait is typically present in significantly larger amounts than its native counterpart in an IP), label-transfer (especially useful for transient interactions), far Westerns, protein arrays, etc.

1. We suggest that you'd better set the positive & negative control for your IP tests and detection.
2. Our advice is to check your IP buffers and detection buffers one by one, if needed.
3. Check the date of your lysates, storage time, freezing and melting record. If the storage time is too long,the protein may have been degradated. When the cell lysates have been freezed and melted several times proteins also may have been degradated.